In one of its life-stages the parasite Schistosoma mansoni (S. mansoni), which causes the tropical disease schistosomiasis, has the unique ability to penetrate intact, unabraded human skin. After gaining entry into the host, the parasite spends over 48 hrs in various layers of the skin without eliciting any marked tissue response. This subdued inflammatory response is largely responsible for the parasite""s ability to pass through the skin virtually undetected by its human host.
Other schistosomes such as Trichobilharia oceltata (a bird parasite) which also can penetrate intact human skin, causes a severe inflammatory response in the skin (commonly called swimmer""s itch) which results in the parasite""s elimination from the host. Therefore, suppression of inflammatory responses in the skin appears to be crucial for the survival of schistosomes in its human host. Because Schistosoma mansoni does not elicit such a response, it would be useful to determine the mechanisms by which the inflammatory response is reduced or suppressed and to exploit such mechanisms as anti-inflammatory therapeutics for treating any inflammatory condition.
The present invention is directed to an isolated purified polypeptide designated Sm 16.8 or variants, fragments, derivatives, homologs or analogs thereof. The variants, fragments, derivatives, homologs or analogs may have a biological activity of Sm 16.8 and/or be immunologically active, that is, capable of inducing antibody formation, the antibodies being directed to one or more epitopes of the Sm 16.8 polypeptide.
Antibodies that specifically bind to polypeptides of the present invention are also within the scope of the present invention. Such antibodies may serve as therapeutics and/or diagnostic products. The antibodies may be polyclonal or monoclonal.
More particularly, the invention is directed to a polypeptide having a molecular weight of 16.8 kDa on a non-reducing SDS-polyacrylamide gel and has an isoelectric point of 5.9. The polypeptide of the present invention is obtainable from the parasite Schistosoma mansoni and may be a secretion/excretion product of the schistosome.
Also, comprehended by the invention are DNAs encoding the polypeptides of the present invention as well as vectors comprising the DNAs of the invention, host cells transformed with the vector and expression products derived therefrom. Host cells may be procaryotic or eucaryotic host cells.
The invention is also directed to vaccines comprising one or more polypeptides according to the present invention which may also include suitable adjuvants, diluents or carrier substances, the vaccines being useful for immunoprophylaxis of schistosomiasis.
Pharmaceutical compositions comprising Sm 16.8 variants, fragments, derivatives, homologs or analogs of the polypeptide in combination with pharmaceutically acceptable diluents, adjuvants and carriers are also contemplated by the present invention.
The invention is further directed to methods of suppressing or preventing inflammation by administering to a subject an anti-inflammatory dose of a polypeptide or pharmaceutical compositions according to the present invention.
Methods for treating inflammation associated diseases such as cutaneous disease are also within the scope of the invention. Such methods comprise administering to a subject a therapeutically effective dose of a polypeptide or pharmaceutical composition according to the present invention. The polypeptides may be administered topically, for example, in a suitable cream, lotion or salve that may include excipients useful in transporting the biologically active polypeptides into the skin.
Methods for treating systemic diseases characterized by inflammatory processes, using the polypeptides of the invention are also contemplated by the present invention. Such diseases may be autoimmune diseases.
During penetration into the skin, parasites such as those of the species Schistosoma continuously excrete/secrete substances (ES) into their surroundings in order to aid their passage and/or as part of their metabolism.
These ES products are known to contain different types of proteins and lipids, many of whose function are not fully known. By culturing these parasites (i.e., schistosomes) in vitro ES products have been collected, purified and analyzed for their functional capabilities. One of the major components of the ES products are proteins. Given that S. mansoni and T. ocellata differ in their ability to induce inflammation in the skin, the proteins in the ES products of the two parasites were analyzed in an attempt to identify whether ES products of S. mansoni contain any factors that have the capacity to modulate inflammatory responses in human skin that are not contained in the ES products of T. ocellata. These studies revealed the presence of a protein in the secretions of S. mansoni but not T. oceltata, that has the ability to suppress inflammation.
Cytokines, natural hormone like substances produced by many cells in the skin, may play a central role in initiating, promoting or suppressing inflammation. Cytokines such as Interleukin-1xcex1(IL-1xcex1) and IL-1xcex2 promote inflammation, whereas, cytokines such as IL-1ra suppress inflammation. In the skin, cells such as keratinocytes, Langerhans cells, lymphocytes or mast cells can produce an array of pro-inflammatory cytokines upon activation. Yet, penetration and migration of S. mansoni through the skin fails to induce any inflammatory, response.
Keratinocytes constitute over 95% of the cells in human skin. It has been shown repeatedly that depending upon the stimulus, human keratinocytes may produce the pro-inflammatory cytokine IL-1xcex1 or the anti-inflammatory cytokine IL-1ra. Production of large quantities of IL-1ra locally results in the suppression of inflammatory responses.
The immunological basis for many cutaneous diseases such as atopic dernatitis, urticaria, contact sensitivity, cutaneous allergic conditions and psoriasis, is the accumulation of inflammatory cells in the epidermis and dermis. Any drug that can reduce or suppress accumulation of inflammatory cells in the skin will alleviate the clinical symptoms associated with these diseases.
Other inflammatory diseases are associated with the inflammatory processes in other organs of the body and are likewise susceptible to treatment with certain anti-inflammatory drugs.
A Schistosoma mansoni derived protein, Sm 16.8, that selectively up regulates IL-1ra production in human keratinocytes was identified and characterized. When added to human keratinocyte cultures at a concentration of 5 xcexcg/1xc3x97105 cells, Sm 16.8 stimulated the production of IL-1ra within 4 hrs. Intracellular message (mRNA) for IL-1ra in these cells increased from 4 hrs after treatment and attained maximum values within 8 hrs. Statistically significant amounts of IL-1ra were also found intracellularly and in the culture supernatants of human keratinocytes after treatment with Sm 16.8. IL-1ra is a natural antagonist of IL-1, in that it competes with IL-1xcex1and IL-1xcex2 for the IL-1 receptor. Binding of IL-1xcex1 or IL-1xcex2 to IL-1 receptors expressed on many cells results in a cascade of events leading to inflammation. However, binding of IL-1ra to IL-1 receptors fails to induce any receptor mediated responses. Therefore, occupancy of all available IL-1 receptors by IL-1ra results in the blocking of all IL-1 mediated responses. Since, IL-1ra binds to IL-1 receptors with affinity equal to or higher than those of IL-1xcex1 or IL-1xcex2 and, since the dissociation rate constant of IL-1ra from IL-1 receptors is several-fold lower than that for IL-1xcex1 and IL-1xcex2, a higher concentration of IL-1ra in the local microenvironment can effectively block all the IL-1 mediated responses including inflammation. Given evidence that Sm 16.8 can stimulate a 100-400 fold increase in IL-1ra production from human keratinocytes, which are the major cell type in the skin, the use of Sm 16.8 as an anti-inflammatory substance against inflammatory diseases including inflammatory conditions in human skin is within the scope of the present invention.
In addition to its effect in stimulating IL-1ra production, Sm 16.8 suppresses IL-1xcex1 and IL-1xcex2 synthesis in human keratinocytes both at the transcriptional and translational levels. When added to human keratinocyte cultures that were stimulated with lipopolysaccharidexe2x80x94a bacterial envelope protein that induces marked IL-1xcex1 production in human keratinocytes, the parasite-derived protein suppressed IL-1xcex1 production by keratinocytes in a dose dependent fashion. At a concentration of 5 xcexcg/ml per 1xc3x97105 cells, Sm 16.8 completely inhibited IL-1xcex1, and IL-1xcex2 RNA production in human keratinocytes. Thus, Sm 16.8 appears to act by providing a two-pronged approach towards reducing inflammation, (i) by stimulating the production of IL-1ra in human skin cells and (ii) by transcriptionally down regulating the production of the pro-inflammatory cytokines IL-1xcex1 and IL-1xcex2.
Lymphocytes collected from infected animals often respond to recall antigens (antigens to which an animal has been previously exposed) by rapid multiplication (lymphoproliferation) and by secreting cytokines, specifically IL-2. This immunological phenomenon provides the basis of many vaccination protocols. It is clearly shown herein that lymphocytes recovered from the spleen and axillary lymphnodes of S. mansoni infected mice exhibit recall response to parasite Schistosoma antigens. However, when Sm 16.8 is present in the antigen mixture, the lymphocytes are unable to respond to the antigens. Once the antigen mixture is depleted of Sm 16.8 the recall response is regained. When Sm 16.8 is then added back to the depleted antigen mixture, ability of the lymphocytes to respond to the recall antigens is again lost. This demonstrates that the anti-inflammatory protein Sm 16.8 also has immunomodulatory functions.
Success of a vaccination protocol largely depends on the initiation of an appropriate immune response at the site of vaccination. In mice a radiation-attenuated vaccine [Richter et al., Parasitology Today, 11:288-293 (1995)] has been shown to confer protection against S. mansoni infection. Such vaccination results in an initial interferon-xcex3 (IFN-xcex3) response in the skin and axillary lymph nodes. Whereas, a normal infection results in an initial IL-4 and IL-10 but no IFN-xcex3 response. Thus, development of an early IFN-xcex3 response in the skin and its associated axillary lymph nodes is correlated with protection. In vitro studies show that Sm 16.8 suppresses IFN-xcex3 response in axillary lymph node cells stimulated with recall antigens. Therefore, it is possible that Sm 16.8 may also interfere with other aspects of the development of immune response against the parasite.
In an important aspect of the invention, antagonists of Sm 16.8 activity such as peptides (the preparation of which are discussed below) and antibodies are produced which, upon challenge with S. mansoni, will block the anti-inflammatory activity/or immunomodulatory effects of Sm 16.8. Antibodies are produced by immunization of a host animal with polypeptides according to the present invention using methods well known in the art.
Another anti-inflammatory protein designated Transthyretin (TTR) [Borish et al., Inflammation, 16(5):471-484 (1992)], has a molecular mass close to the parasite-derived protein Sm 16.8 of the present invention. TTR has a molecular mass of 17 kDa and is produced by human liver cells. However, studies show that TTR is different from Sm 16.8 both in its physical property and function. TTR was originally purified from human serum by an anion exchange chromatography, molecular sieve HPLC and hydroxyl apatite chromatography suggesting that TTR is a basic protein. Whereas Sm 16.8 is an acidic protein with a pI of 5.8. [See, e.g., Borish et al., Inflamination, 16(5):471-484 (1992); and Zocchi et al., Immunol. Letters, 13(5):245-253 (1986).]
The anti-inflammatory activity of TTR depends on its ability to inhibit secretion of IL-1xcex1 and IL-1xcex2 from endothelial cells and monocytes similar to Sm 16.8. However, TTR does not inhibit the intracellular synthesis of IL-1 as does Sm 16.8, rather TTR increases the level of IL-1 mRNA and IL-1 protein concentration intracellularly. This means the TTR treated cells are continually synthesizing the pro-inflammatory cytokine IL-1 in their cytoplasm but are unable to secrete the cytokine, whereas, in Sm 16.8 treated cells the synthesis of pro-inflammatory cytokines IL-1xcex1 and IL-1xcex2 is completely inhibited. In addition, unlike TTR, Sm 16.8 is shown to stimulate the production and secretion of significant quantities of the anti-inflammatory cytokine IL-1ra. Thus overall, the function of Sm 16.8 provides advantages in reducing an inflammatory response in the skin not obtainable with TTR because it reduces or eliminates the pro-inflammatory cytokines present in the microenvironment of the tissue.
Having isolated and purified Sm 16.8 its amino acid composition and its N-terminal amino acid sequence are obtainable using routine methodologies well known to those of ordinary skill in the art. Knowledge of the N-terminal amino acid sequence of Sm 16.8 enables a person of ordinary skill in the art to obtain the DNA encoding the protein by synthesizing appropriate polynucleotide probes or primers based on the determined amino acid sequence and by using the polynucleotides or probes to screen genomic DNA libraries or cDNA libraries obtainable from S. mansoni by hybridization methods, or by use of methods such as the polymerase chain reaction (PCR). Upon isolating and determining the sequence of a DNA encoding Sm 16.8, a person of ordinary skill in the art having knowledge of the genetic code would be readily able to deduce the complete amino acid sequence of Sm 16.8. Vectors comprising a DNA according to the present invention (e.g., plasmids, viruses, bacteriophage, cosmids and others) are useful for expressing the Sm 16.8 DNA in host cells. Host cells may be eucaryotic or procaryotic cells. Such cells include yeast cells and bacterial cells such as E. coli and others. Host cells expressing DNAs of the present invention provide an abundant reproducible source of Sm 16.8 for use in the practice of the invention.
DNA of the present invention are also useful for preparing muteins, variants or analogs of Sm 16.8, having amino acid substitutions at specified sites in the protein and which retain a biological activity of Sm 16.8. Methods for selecting candidate amino acids for substitution are well known in the art. [See, e.g., Kyte et al., J. Mol. Biol., 157:105-132 (1982).] The DNA may also be used to prepare truncations or fragments of Sm 16.8 and to prepare derivatives of Sm 16.8 such as Sm 16.8 fusion proteins. Exemplary proteins for fusion to Sm 16.8 include xcex2-galactosidase, glutathione-S-transferase, (His-tags) and others well known in the art. The fusion may be made at the N-terminus, carboxyl terminus of the polypeptide or may be inserted at an internal site of the protein. Such fusion proteins may be useful in preparing vaccines for enhancing the immune response directed to Sm 16.8 and thereby serving as an immunoprophylactic against infection with S. mansoni. The vaccine may also comprise suitable diluents, adjuvants, and carriers.
Useful fragments of Sm 16.8 may also be prepared by the proteolytic digestion of the purified protein with one or more of a variety of well known proteolytic reagents such as cyanogen bromide and/or proteolytic enzymes such as the carboxypeptidases, asparaginases and others well known in the art.
DNAs, according to the present invention, may also be used to isolate DNA homologous from other species by hybridization at high stringency or by PCR under stringent conditions. Molecular techniques for accomplishing the foregoing are well known and described in detail in numerous publications including Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons (1996), which is incorporated herein by reference.
Polypeptides of the present invention may also be covalently modified by the addition of chemical moieties. Exemplary chemical moieties include polyethylene glycol.
Once the primary amino acid composition of the protein is obtained the three-dimensional structure of the protein is determined, e.g., by crystallization and x-ray diffraction, and the active binding sites could be identified for specific therapeutic applications or immunomodulation. [See generally, e.g., PCT/U.S. Ser. No. 93/05548 published Dec. 23, 1993.] Such therapeutics may be small peptides or polypeptide fragments that bind to the active site thereby blocking its activity or may be other small molecules which interfere with the active site of the polypeptide.